CN1069322C - Preparation and usage of antisense digonucleotides - Google Patents
Preparation and usage of antisense digonucleotides Download PDFInfo
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- CN1069322C CN1069322C CN97120355A CN97120355A CN1069322C CN 1069322 C CN1069322 C CN 1069322C CN 97120355 A CN97120355 A CN 97120355A CN 97120355 A CN97120355 A CN 97120355A CN 1069322 C CN1069322 C CN 1069322C
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Abstract
The present invention relates to a biological engineering product and the purpose thereof, more specifically two antisense oligonucleotides which are designed and prepared according to influenza virus genomes and are used for inhibiting influenza virus duplication. Aiming at the conserved sequences of 3'UCGU/CUUUCGUCC5' and 3'GGAACAAAGAUGA5' at the 3' end and the 5' end of A type influenza virus genomes, the present invention firstly discovers, designs and synthesizes complementary oligonucleotides (ODN) of IV3#ODN(5'AGCI<*>AAAGCAGG3') and IV4#ODN(5'CCTTGTTTCTACT3'), wherein I<*> represents hypoxanthine basic groups. The oligonucleotides are connected by phosphate ester bonds with the molecular formula of-O-P(=O)(Y)-O-; in the molecular formula, Y represents oxygen, sulphur or methyl, etc.; both of the sulfo objects and the aliphatic chain decorative objects of the oligonucleotides can effectively inhibit the duplication of influenza viruses in MDCK cells under the action of liposomes, fusogenic peptides, etc.; the inhibiting effect presents sequence and using quantity dependency; in mouse models infected by influenza viruses, IV4#ODN can effectively inhibit lung pathological changes caused by influenza virus infection and reduce the lung influenza virus titer of infected mice; further, the new purpose of IV3#ODN, IV4#ODN and the decorative objects thereof for resisting influenza virus infection is discovered. The biological engineering product of the present invention is a new biological engineering medicine used for treating influenza.
Description
The present invention relates to the biotechnology medicine, relate in particular to the preparation of the few nucleic acid drug of antisense and the purposes of control influenza virus.
Influenza virus is the main pathogen of respiratory tract infection.Because influenza virus variation, conventional vaccine such as inactivated vaccine and attenuated live vaccines effectively flu-prevention take place with popular.The treatment influenza medicine Buddha's warrior attendant amine of present unique input clinical application has neurotoxic side effects, easily causes defectives such as resistance strain appearance, therefore study the specificity height, new type influenza medicine that toxic action is little has important realistic meaning (Nicholson et al.Seminars in Respiratory Infection 1992,7 (1): 26-37 to treatment of influenza and prevention; Kimberlin et al.Antiviral Res.1995,26 (4): 369).Antisense oligonucleotide (Antisense Oligonucleotide, ASON) be that a class can combine the also synthetic DNA segment of specific inhibition genetic expression with DNA/RNA, be potential newtype drug (the Stein et al.Science 1993,262 (20): 1004-1012) of treatment viral infectious and tumour.Having attempted utilizing antisense oligonucleotide to suppress influenza virus abroad duplicates.Zerial in 1987 etc. screen ODN (Zerial et al.Nucleic Acids Res.1987,15 (23): 9909-9919), but need further research as medicinal specificity be made up of 7 Nucleotide that can suppress that A type influenza virus duplicates.It is ODN (the kabanov et al.FEBS Letters 1990,259 (2): 327-330 that target spot screens anti-specific influenza virus strain with genes such as A type influenza virus PA, PB1, PB2, NP that report is also arranged; Leiter et al.PNAS 1990,87 (9): 3430-3434; Hata et al.Biochem Biophys Res Commun 1996,223 (2): 341-346; 1997,232 (2): 545-549).Consider that the action target spot sequence guards degree, the broad spectrum of these ODN anti-influenza type A virus remains further to be confirmed.
The objective of the invention is according to influenza virus gene group design and prepare two oligonucleotide, can be used as the medicine of influenza infection with this.
To the effect that of the present invention, with each segment of A type influenza virus gene group 5 ' end (5 ' AGUAGAAACAAGG3 ') and 3 ' end (5 ' CCUGCUUUU/CGCU3 ') conserved sequence is target spot, design and synthesize with 5 ' end complementary ODNIV4# (5 ' CCTTGTTTCTACT3 ') and with 3 ' hold complementary ODNIV3# (5 ' AGCI
*AAAGCAGG3 '), with A type influenza virus representative strains A1/ capital anti-/ 86-1 (H1N1) and A3/ Shandong prevent/93-9 (H3N2) is an object, with hirst's hemagglutination (HA) titre and cytopathic effect (CPE) is index, IV4# and IV3# inhibition influenza virus duplication characteristic in mdck cell system, have been estimated, the result shows that IV3# and IV4# can duplicate by specificity inhibition influenza virus.For example, when IV4# concentration is 1 μ mol/L to viral inhibiting rate nearly 50%; Concentration is 10 μ mol/L or the virus replication inhibiting rate is surpassed 70% when higher.And ODN suppresses virus activity and is sequence and dose-dependently.In the influenza infection mouse model, IV4# and IV3# can effectively suppress the tuberculosis change that influenza infection causes, and reduce virus titer in the lung.In a word, according to the present invention, duplicating by specificity inhibition influenza virus with A type influenza virus gene group 5 ' end and the complementary bonded oligonucleotide IV3# of 3 ' end conserved sequence and IV4#, is a kind of novel biological engineering medicine that is applied to treat with the flu-prevention virus infection.
About the length of oligonucleotide IV3# and IV4#, the invention discloses IV3# length is 12 Nucleotide, and IV4# length is 13 Nucleotide.The length of antisense oligonucleotide and its cell permeability, relevant with factors such as target sequence binding affinity, effect specificitys.The shortest length of IV3# and IV4# needs to determine that according to experiment the present invention has comprised any length oligonucleotide that has identical sequence with IV3# and IV4#.
The antisense oligonucleotide that suppresses influenza virus can increase its stability by modifying.Forming the bound phosphate groups of oligonucleotide can modify in the free singlet oxygen position of natural phosphodiester key part, wherein can be oxygen, methyl or sulphur.During preparation, solid support is connected, the oligonucleotide deprotection that contains nucleotide sequence of the present invention that passes through solid phase synthesis of protection, cuts off from solid support.Disclosed by the invention promptly is sulfo-IV3# and the IV4# for preparing by this method.Therefore the preparation and the modifying method thereof of oligonucleotide also are one of contents among the present invention.
According to invention, be used to prevent and treat the antisense oligonucleotide composition of influenza infection, wherein contain at least a oligonucleotide of the present invention or its modifier; If suitably, can also contain treatment and go up the acceptable carrier material, as liposome, fusogenic peptide or virus vector etc.
Accompanying drawings specific implementation method of the present invention is as follows:
Fig. 1 be the ODN IV3S that constitutes of IV3#ODN, IV4#ODN and just sequence thereof and IV4S to influenza virus A/ capital anti-/ restraining effect that 86-1 (H1N1) duplicates in MDCK.Fig. 2 be IV4#ODN to influenza virus A/ capital anti-/ restraining effect of 86-1 (H1N1) cytopathic effect in MDCK.Wherein, normal mdck cell Fig. 3 of A. influenza infection MDCK B. influenza virus+0.1 μ mol/L ODN C. influenza virus+1 μ mol/L ODN D. influenza virus+10 μ mol/LODN E. uninfecting virus for IV4#ODN to influenza virus A/ Shandong anti-/ restraining effect that 93-9 (H3N2) and A/ Shandong/93-9 (H3N2) duplicate in MDCK.Fig. 4 prevents/restraining effect that 86-1 (H1N1) duplicates in MDCK influenza virus A/ capital down in liposome (Lipofectin) effect for IV4#ODN and aliphatic chain modifier IV4#R thereof.
Embodiment one: the technical essential of present embodiment is as follows:
1.MDCK clone and influenza virus A/capital is anti-/ and 86-1 (H1N1), A/ Shandong be anti-/ 93-9 (H3N2) and A/ Shandong/93-3 (H3N2): the MDCK nutrient solution uses when propagative viruses and ODN screening to contain the tryptic DMEM of 5g/ml for containing the DMEM nutrient solution of 10% calf serum (GIBCO.BRL).The freeze-drying influenza virus is gone down to posterity 3-4 time at 10 age in days SPF chicken embryos, collects allantoic fluid, and 2, centrifugal 10 minutes of 000g, packing ,-70 ℃ of preservations are as the virus stock solution used that infects MDCK.
2. sulfo-dna oligo (PS-ODN) design is with synthetic: with reference to A type influenza virus gene group end conserved sequence, design four ODN:IV3#:5 ' AGCI
*AAAGCAGG3; IV3S:5 ' CCTGCTTTIGCT3 ' IV4#:5 ' CCTTGTTTCTACT3 ' IV4S:5 ' AGTAGAAACAAGG3 '.PS-ODN is synthetic at Applied Biosystems 391DNA synthesizer.Synthetic back is used 55 ℃ of deprotections of strong aqua 15 hours earlier, uses reversed-phase column (available from Solid PhaseSciences company) purification by chromatography then.
3. resisiting influenza virus antisense oligonucleotide screening: with 10
6Individual cells/well is inoculated into 96 porocyte culture plates (Nunc) to MDCK, and cell growth medium is the DMEM that contains 10% calf serum.37 ℃ of overnight incubation are with PBS (pH7.5) flushing 3 times.With the DMEM that contains 0.2%BSA PS-ODN is diluted to following concentration: 0.1 μ mol/L, 1 μ mol/L, 5 μ mol/L, 10 μ mol/L, 20 μ mol/L add each hole (each extent of dilution 4 hole) with the amount in 50 μ l/ holes, 37 ℃ of effects 1 hour; Again with PBS (pH7.5) flushing 3 times, the influenza virus A/capital that adds 50 μ l 320 * dilution is anti-/ 86-1 (H1N1) (MOl=1), 37 ℃ act on 45-60min; With PBS (pH7.5) flushing 3 times, add the DMEM that contains 0.2%BSA, 5 μ g/ml trypsinase and same concentrations PS-ODN, set up virus infection positive control and MDCK negative control simultaneously.Cultivated 2 days for 37 ℃, suppress the virus replication activity by effect of observation of cell pathological changes caused by virus and viral hemoagglutination titer determination method evaluation PS-ODN.
Fig. 1 be PS-ODN IV4#, IV4S, IV3#, IV3S to influenza virus A/ capital anti-/ blood clotting titre inhibiting rate that 86-1 (H1N1) duplicates at mdck cell.As seen from the figure, when concentration was 1 μ mol/L, 5 μ mol/L, 10 μ mol/L, 20 μ mol/L, IV4## all had the inhibition virus activity.When concentration is 1 μ mol/L, to viral HA titre inhibiting rate nearly 50%; When concentration is 10 μ mol/L or when higher, the virus replication inhibiting rate is surpassed 70%; And the restraining effect that IV4# duplicates influenza virus is concentration dependent.IV4S concentration does not show when being 1 μ mol/L, 5 μ mol/L and suppresses active, and slight inhibition virus activity is arranged when high density, and inhibiting rate is less than 50% when being 20 μ mol/L as concentration.IV3# is similar substantially to IV4S to the effect of influenza virus, but concentration is when being 1 μ mol/L, 5 μ mol/L, and the IV3# performance promotes the virus replication effect, when being 1 μ mol/L as concentration, makes virus titer raise 50%.IV3S concentration does not have the inhibition virus activity when being 1 μ mol/L, concentration only shows slight restraining effect when being 5 μ mol/L, 10 μ mol/L, 20 μ mol/L.On the other hand, IV4# suppresses viral CPE effect, and the big more restraining effect of concentration more obvious (Fig. 2).Above result shows that IV4#PS-ODN has the duplicate effect of the influenza virus of inhibition in MDCK, and the inhibition virus activity is sequence-specific and dose-dependently.Simultaneously, discovery IV4#ODN suppresses the A/ Shandong equally, and anti-/ 93-9 (H3N2) and A/ Shandong/93-9 (H3N2) duplicate (Fig. 3) in MDCK, show that IV4#ODN can specificity suppress duplicating of A type influenza virus representative strains.4. liposome Lipofectin suppresses the influence of virus activity to IV4#PS-ODN: the mdck cell that grows up to the 70-80% individual layer on the 96 porocyte culture plates, with PBS (pH7.5) flushing 3 times, every hole adds the DMEM100 μ l that contains S-ODN (concentration is respectively 0.1 μ mol/L, 1 μ mol/L, 10 μ mol/L) and 0.4 μ l (1 μ g/ μ l) lipofectin (GIBCO.BRL), 37 ℃ act on 4 hours, and then the method evaluation ODN by this technical application main points (3) suppresses virus activity.
Fig. 4 is that IV4#ODN and aliphatic chain modifier thereof are under the lipofectin transfection conditions during to the inhibiting rate of hirst's hemagglutination titre, no lipofectin, IV4#R is not so good as IV4# to viral inhibition, when being 1 μ mol/L and 10 μ mol/L as concentration, the inhibiting rate of IV4# is 56.2% and 78.1%, and the inhibiting rate of IV4#R is 30% and 50%.Under the lipofectin transfection conditions, IV4# and IV4#R suppress virus activity obviously to be strengthened, and inhibiting rate is respectively 31.2%, 75% and 60%, 77.5% when being 0.1 μ mol/L, 10 μ mol/L as concentration.IV4#ODN and its aliphatic chain modifier IV4#R suppress virus activity difference under the lipofectin transfection conditions not obvious.IV4# and IV4#R suppress virus activity when the lipofectin transfection close with the virofral action effect, and virofral is respectively 79.7% and 100% to viral inhibiting rate when concentration is 0.5 μ mol/L and 5 μ mol/L.
Embodiment two: the technical essential of present embodiment is as follows:
1. influenza virus adapts to the mouse process: be taken at the A/ capital that 11 age in days SPF chicken embryos go down to posterity anti-/ (the blood clotting titre is 2 to 86-1 (H1N1)
9HAU/40 μ l) 50 μ l, by the collunarium mode inoculate 6 week BALB/C in age female mouse, raises get under the sterile state after 3 days lung with PBS with Wt/V=1: 10 prepare lung homogenate,-20 ℃/37 ℃ freeze thawing three times, 5, centrifugal 10 minutes of 000rpm gets supernatant and goes down to posterity mouse with the same manner.Go down to posterity altogether 6 times, get the lung homogenate supernatant at last and inoculate 11 age in days SPF chicken embryos, measure the HA titre, packing ,-70 ℃ of preservations are standby.
2. antisense oligonucleotide suppresses the influenza virus activity rating in mouse model: get mouse and adapt to influenza virus A 1M6 (29HAU/40ul) 50 μ l, inoculate the female mouse of 6 week BALB/C in age by the collunarium mode, inoculate antisense oligonucleotide IV4# by the dosage of 100 μ g/ mouse with the same manner simultaneously, every day by the dose inoculations of 100 μ g/ mouse once then, inoculate altogether 5 times, raised 2 days after the drug withdrawal, weigh, get lung and weigh, with Wt/V=1: 10 preparation lung tissue homogenate, virus titer is measured in-20 ℃/37 ℃ freeze thawing three times in MDCK.Find that antisense oligonucleotide IV4#ODN can obviously stop the mouse body weight to reduce, inoculation IV4#ODN mouse lung weight/weight average value is near normal mice, and can suppress obviously that tuberculosis that influenza virus causes becomes and lung in virus titer, illustrate that antisense oligonucleotide IV4#ODN can effectively suppress duplicating of influenza virus in the mouse model.Can use the influenza infection treatment.
Claims (2)
1. comprise two oligonucleotide of being made up of specific nucleotide sequence, it is characterized in that, a sequence is 5 ' AGCI
*AAAGCAGG3 ', code name are IV3#ODN, wherein I
*Represent the xanthoglobulin base; Another sequence is 5 ' CCTTGTTTCTACT3 ', and code name is IV4#ODN.
2. the oligonucleotide of claim 1 is in the purposes of preparation in the anti-influenza virus medicament.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US8357664B2 (en) * | 2004-10-26 | 2013-01-22 | Avi Biopharma, Inc. | Antisense antiviral compound and method for treating influenza viral infection |
| CN101792762B (en) * | 2007-04-20 | 2012-04-18 | 清华大学深圳研究生院 | Antisense oligonucleotide for specifically adjusting POKEMON gene expression and application thereof |
| CN102295673A (en) * | 2011-07-07 | 2011-12-28 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Structure, preparation method and application of oligonucleotide modified by aliphatic chains |
| CN102351932A (en) * | 2011-07-07 | 2012-02-15 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Structure, preparation method and purpose of anti-influenza virus oligonucleotide |
| CN113502288B (en) * | 2020-07-23 | 2023-05-09 | 纳肽得(青岛)生物医药有限公司 | Antisense oligonucleotides and their use in inhibiting novel coronaviruses |
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| CN1131437A (en) * | 1993-07-27 | 1996-09-18 | 海布里顿公司 | Antisense oligonucleotide inhibition of vascular endothelial growth factor expression |
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| CN1131437A (en) * | 1993-07-27 | 1996-09-18 | 海布里顿公司 | Antisense oligonucleotide inhibition of vascular endothelial growth factor expression |
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